The present invention relates to agglutination assays, and especially to such assays which quantitate binding pair members in a sample through the use of a light transmission or scattering measurement.
Agglutination immunoassays are known wherein particles coated with an immuological binding pair member (antigen or antibody) agglutinate in the presence of active complementary binding pair members. In the simplest such immunoassays, the level of antigen in the sample affects the extent of agglutination of antibody-coated particles, with increased turbitity being caused by increased agglutination, as an indication of elevated levels of target antigen. In competitive agglutination immunoassays, a fixed amount of antibody in solution causes reduced levels of agglutination of antigen-coated particles as increased levels of target antigen compete for limited binding sites on the antibody. Conventionally, such agglutination is determined qualitatively by visual observation after a fixed reaction period, in some cases after rapid centrifugation at the conclusion of the reaction period to concentrate the agglutinated particles as in U.S. Pat. No. 4,373,931 to Takekawa (1983).
Quantitative analysis of agglutination by light transmission or scattering measurement has been proposed in a variety of patents: e.g., U.S. Pat. Nos. 4,118,192 to Sawai et al. (1978), 4,205,954 to Babson (1980) and 4,224,304 to Sawai et al. (1980). In these patents, a plurality of light transmittance measurements are taken over time; and the change in transmission, due to the change in optical density corresponding to agglutination of single particle into clusters, is used as an indication of the degree of agglutination. Such measurements rely upon a constant total particle concentration in the sample light path, with the local rearrangement of particles in clusters of two or more particles affecting light differently over time as a function of agglutination.
U.S. Pat. No. 4,202,665 to Wenz (1980) and Wenz et al., Clin. Chem. 25: 1613-1616 (1979) disclose a hemagglutination assay for hepatitis performed in a microcentrifugal analyzer. Rapid changes in absorbance due to centrifugation are observed (over a 90 second time frame in most cases); the slope of absorbance decline is increased by agglutination of the antibody-coated cells when antigen is present. Other types of particles, including polymers, are referred to.